The purpose of this project is to develop methods to effectively immunize human cells in vitro against tumor antigens (or model antigens in well-documented systems), and to use these cells as fusion partners to produce human-human or human-mouse hybridomas secreting antitumor antibody. During this year, we have studied patients being treated in the anti-melanoma monoclonal antibody clinical trial conducted by the Clinical Investigations Section, BTB, BRMP, DCT. Various strategies of antigen presentation, timing, and culture modifications were evaluated. The first four patients treated with the 9.2.27 murine anti-melanoma antibody were evaluated prior to and following antibody therapy. None of the patients' peripheral blood lymphoid cells produced specific anti-melanoma antibody. However, lymphocytes obtained following therapy could be stimulated to produce anti-M14 antibody by incubating these cells in vitro for 8-14 days in the presence of a solid-phase complex of melanoma cell (M14) extract presented on 9.2.27 antibody. This was true in all four of the patients studied thus far. Recently, human-human hybridoma fusions were performed using such in vitro immunized cells and the fusion parent line HF-2. Although some human B-B hybridomas have been found to produce anti-melanoma antibody, the hybridoma cultures are not yet cloned. The oldest hybridoma culture is 80 days old (May, 1983).